Labelled boronic acid derivatives

ABSTRACT

The invention provides novel labelled boronic acid conjugates of formula ##STR1## (wherein V is a reporter moiety; W 2  is a bond or an organic linker moiety; 
     W 1  is a *SO 2  NR 2 , *CON 2  or *CH 2  N⊕R 2   2  group bound at the *-marked atom to the phenyl ring; 
     R 1  is hydrogen or an electron withdrawing substituent group; and 
     each R 2  independently is hydrogen or an optionally hydroxylated and optionally C 1-6  -alkoxylated C 1-6  -alkyl group) and salts thereof, e.g. for use in assays for cis-diols such as glycated blood proteins, having enhanced water-solubility and storage stability.

FIELD OF THE INVENTION

The present invention relates to novel boronic acid derivatives carryinga reporter species or "label".

BACKGROUND OF THE INVENTION

Particularly in diagnostic medicine it is well established practice touse labelled species to bind to substances of interest (analytes) inorder that the intensity of the original from the lable may be used toascertain the presence or concentration of the analyte.

Such labelled species require two functional attributes--the first beingthe ability to produce a detectable signal, actively or passively, andthe second being the ability to bind to a desired binding partner.

The signal forming attribute may conveniently be fulfilled by thepresence in the labelled species of a reporter moiety or "label" whichitself acts as a radiation emitter, absorber or modifier or whichcooperates with another species to achieve a radiation emitting,absorbing or modifying effect. Thus for example the label may be achromophore, a fluorophore, a radionuclide, or a material havingdetectable magnetic characteristics, e.g. paramagnetism,superparamagnetism etc.

One form of labelled species that has been proposed for diagnostic use,e.g. in assay lists, fulfills the binding ability attribute by theinclusion in the species a --B(OH)₂ or --B(OH)3--binding group.

Such boronic acid conjugates have the ability to bind to cis-diols, e.g.in proteins. By way of example, boronic acid conjugates of this natureare described in WO-92/08722, US-A-4659817, DE-A-3720736 andUS-A-4861728. The compounds described there comprise antibody orchromophoric or fluorophoric label moieties. Such compounds are usefulin assays for glycated blood proteins in view of their ability to bindto the glycosyl moieties. The labelled conjugates of WO-92/08722 areespecially useful in this regard as the chromophores and fluorophoresthere described have absorption maxima above 600 nm and thus there is nointerference between their spectral response and that of hemoglobin.

These labelled boronic acid conjugates can be represented by the generalformula I

    V-W-B(OH).sub.2                                            (I)

where V is a reporter moiety, e.g. a chromophore, fluorophore orradioisotope and W is a bond or linking organic group.

WO-92/08722, like other publications describing labelled boronic acidconjugates, suggested that the linker group, W in formula I above, mightsuitably incorporate a m-aminophenyl group attached to the boronic acidresidue. Thus such compounds may readily be prepared by conjugating thelabel to m-aminophenyl-boronic acid thereby producing a compound offormula II ##STR2##

(where V is as defined above and W² is a bond or an organic linkermoiety).

Such compounds however have been found to have limited acceptability interms of stability and water solubility and for certain assay procedureshave necessitated the use of organic co-solvents such as DMSO, DMF andformamide rather than of water alone.

SUMMARY OF THE INVENTION

It has now been found that by replacement of thecarbonylamino-1,3-phenylene linker group previously used, labelledboronic acid conjugates having improved stability and improved watersolubility may be obtained.

Thus viewed from one aspect the invention provides labelled boronic acidconjugates of formula III ##STR3## (where V is a reporter moiety; is abond or an organic linker moiety;

W¹ is a *SO₂ N², *CON², or, less favorably, a *CH₂ N⊕² ₂ group attachedat the * marked atom to the phenyl ring;

R¹ is an electron withdrawing functional group, e.g. a SO₂ H, COOH orNO₂ group; and

each R² independently is hydrogen or optionally hydroxylated, optionallyC₁₋₆ -alkoxylated C₁₋₆ -alkyl) and salts thereof.

DETAILED DESCRIPTION

The new compounds have reduced pKa, better water-solubility andincreased stability in aqueous solution than the analogousm-aminophenyl-boronic acid conjugates.

Since it is generally accepted that it is the anionic form of theboronic acid that participates in the cis-diol esterification, the lowerpKa is advantageous insofar as it permits a lower pH to be used in thecoupling reaction with the cis-diol analyte while maintaining asufficiently high concentration of the dissociated boronic acid. This isparticularly advantageous in blood assays where zinc ions are used toprecipitate hemoglobin due to the increased stability of the zinc ionsat the lower pH. At alkaline pH's zinc has a tendency to produceunwanted insoluble hydration complexes.

Previously this problem has had to be addressed by inclusion of furthercomplexing agents which stabilize the zinc until the hemoglobin isadded. Such agents must of course form weaker complexes with zinc thandoes hemoglobin itself in order to allow the zinc to effect hemoglobinprecipitation. Thus, the reduction of the pH of the solution of labelledboronic acid and zinc, ideally to about 7 to 8, is made possible by thelower pKa of the boronic acid and facilitates the balance of complexingagents needed to stabilize the zinc.

To enhance pKa reduction and improve boronic acid stability in thecompounds of formula III, it is especially desirable to have the phenylring substituted by one or more electron withdrawing substituents R¹.Most effectively such substitution is at the ortho or para positionsrelative to the boronic acid residue. Moreover in those compounds of theinvention where W¹ is attached to the phenyl ring via a heteroatom or anunsaturated carbon, attachment is desirably at the para position whilefor the CH₂ attached moieties attachment is preferably at the meta orpara positions.

The precise chemical nature of the VW² moiety is of secondaryimportance--its primary function is simply to provide a reporter moiety(i.e. a label) and means for attaching that label to the W¹ moiety.

Nonetheless, certain W² linker moieties have been found to beparticularly suitable for use in this regard and in this context mentionmay be made of optionally substituted aza and/or oxa-alkylene groups,e.g. groups of formula IV

    --X.sup.1 --[(CHR.sup.3).sub.n --X.sup.2 ].sub.m --(CHR.sup.3).sub.n --IV

(wherein each n is independently an integer of 1 to 6 preferably 2 or 3,especially 2;

m is 0 or a positive integer, e.g. 1 to 5, m preferably being 0, 1 or 2;

X¹ is a bond, an oxygen or sulphur atom, a NR² group, or a carboxy orcarbonyl group or the residue of any other functional group serving tolink W² to V, X¹ preferably being a group NR² ;

each X² is independently an oxygen or sulphur atom or an NR² group, X²preferably being O or N² ;

each R³ independently is hydrogen, hydroxy, formyl, carboxy, oroptionally hydroxylated and/or C₁₋₆ -alkoxylated C₁₋₆ -alkyl, or a groupCHR³ may represent a carbonyl group, however is preferably hydrogen onall but one of the carbons of any (CH³)_(n) moiety, being hydrogen,hydroxy or carboxy on the remaining carbons; and each alkyl or alkylenemoiety, unless otherwise stated conveniently contains 1 to 6, especially1 to 3 carbons) optionally in anionic, cationic, or zwitterionic form.In such a linker moiety the (CH³)_(n) moiety remote from X¹ is attachedto the W¹ moiety and the group X¹ is coupled to the label V.

Examples of such groups of formula IV include but are not limited to thefollowing:

--NH--CH₂ --CHOH--CH₂ --

--NH--CH₂ CH₂ --NH--CH₂ CH₂ --

--NH--CH₂ CH₂ --O--CH₂ CH₂ --

--NH--CH₂ CH₂ --

--NH--CH₂ CH₂ CH₂ --

--NH--CH₂ CH₂ CH₂ CH₂ --

--NH--CH₂ CH₂ CH₂ CH (COOH) --

--NH--CH₂ CHOH--

--NH--CH₂ CH(COOH)--

--NH--CH₂ --NH--CH₂ --

--NH--CH₂ CH₂ CH₂ CH₂ CH₂ CH₂ --

--NH--CH₂ CH₂ CH₂ CH₂ CH₂ --

--NH--CH₂ --O--CH₂ --

--NH--CH₂ --O--CH₂ --O--CH₂ ----NH--CH₂ CH₂ --O--CH₂ CH₂ --O--CH₂ CH₂ --

--NH--CH (COOH) CH₂ CH₂ --

--NH--CH (COOH) CH₂ CH₂ CH₂ --

--NH--CH₂ CH(COOH)CH(COOH)--

--NH--CH₂ CHOHCHOH-- and

NH--CH₂ CH(CHO)CH(CHO) --

However other spacer groups having functional groups appropriate forattachment to label moieties, e.g. nitrile, halide, epoxide, carbonyl,carboxyl, amine, phosphonate and sulphonyl groups may of course be used,especially where they also serve to enhance water solubility.

The reporter moiety, as mentioned above, may be any moiety capable ofproducing an assessable signal. Generally however chromophores andfluorophores, e.g. azine, triarylmethine, phthalocyanine and cyaninedyes, will be preferred for reasons of ease of assessment, safety,efficacy and expense. Particularly preferred for labelled agents for usein blood assays will be compounds in which the chromophore orfluorophore has an absorption maximum in the range 600-1000 nm,especially those triphenylmethine dyes which have this characteristic.

In preferred embodiments, the conjugates of the invention are offormulae V, VI or VII ##STR4##

(where X³ is CO or SO₂ ;

W³ is NHCO or SO₂ NH; and R¹ is hydrogen or, preferably, an electronwithdrawing group, e.g. SO₂ H, COOH or NO₂) and salts and complexesthereof.

In formulae V to VII, linker groups W² are preferably of formula IV andespecially preferably carry at least one solubilizing (hydrophilic)group. In the compounds of formula VI, the R¹ group is preferably in anortho or para position and the VW² W³ group in a meta or para position,in each case relative to the B(OH)₂ group.

The labelled compounds of the invention may be prepared by coupling areporter moiety to a substituted phenylboronic acid optionally using afurther reagent to provide the whole or part of the linker group betweenthe phenylboronic acid and the reporter moiety. Particularlyconveniently however, the labelled compounds of the invention areproduced by coupling a reporter molecule to a linker-substitutedphenylboronic acid of formula VIII ##STR5##

(where X², R¹, R³, n, m and W¹ are as defined above and X¹ is afunctional group reactive to couple to a reporter molecule, e.g. aleaving group such as a halide, or an acid or activated acid group, suchas a carboxyl group, or an amine, hydroxyl or thiol group). Thecompounds of formula VIII and salts thereof form a further aspect of theinvention.

The coupling of the reporter molecule to phenylboronic acids can beachieved using conventional chemical techniques starting from reagentswhich are commercially available or are known from the literature.Similarly the compounds of formula VIII may be prepared by conventionalchemical techniques starting from such known compounds asaminophenylboronic acids, methylphenylboronic acids etc. Exemplaryreaction schemes are set out below: ##STR6##

In the reaction scheme set out above, the reporter molecule coupled inthe production of compounds 12 to 18 is a cyanine dye. While for thepurposes of glycated blood protein assays such dyes are preferred,especially where they have fluorescence absorption maxima above 600 nm,other reporter moieties may be used particularly for other assayprocedures. Examples of suitable reporter moieties are described forexample by Axis Biochemicals AS in WO-92/08722, by Gallop inUS-A-4496722, by Wagner in US-A-4861728, by Schleicher in DE-A-3720736,by Pease in US-A-4830786.

Particularly preferred are triarylmethine dye, e.g. a triphenylmethinedye such as those of formula IX ##STR7##

in which each R₁ independently represents a hydrogen atom, an organicgroup or a (preferably hydrophilic) substituting group (e.g. hydroxy,carboxy, sulpho or chlorosulphonyl), and at least two of the A groupsrepresent auxochrome groups, any non-auxochrome A group being as definedfor R₁. Preferred auxochrome groups include amine/imine systems asrepresented by N(R)₂ /N⁺ (R)₂ and NHR/N⁺ HR systems (where each Rrepresents an organic group, e.g. as described below for R₁, preferablya lower alkyl group such as methyl or ethyl), alone or in combinationwith O/OH. The linker-phenylboronic acid moiety may, for example, beattached to one of the phenyl rings, e.g. coupled via a group such ascarboxy or sulpho, or to one of the R groups in an amine/imineauxochrome system. It will be appreciated that the other resonancestructures and enantiomers which may be drawn are also intended to bewithin the scope of formula IX.

Where any R₁ represents an organic group this may, for example, beselected from alkyl, alkenyl and alkynyl groups, e.g. containing up to 6carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, t-butyl, vinyl, allyl, ethynyl or propargyl; cycloalkyl orcycloalkenyl, e.g. containing 5-7 carbon atoms, such as cyclopentyl,cyclohexyl, cycloheptyl, cyclohexenyl or cyclopentadienyl; aryl groups,e.g. containing 6-12 carbon atoms, such as phenyl, tolyl or naphthyl;heterocyclic rings, e.g. 5-7 membered saturated and unsaturated ringscontaining at least one heteroatom selected from oxygen, nitrogen andsulphur, such as furyl, thienyl, pyridyl, pyrimidyl, pyridazyl,thiazolyl, thiazinyl, triazinyl, pyrrolyl, imidazolyl, pyrazolyl,pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl; lower (e.g. C₁₋₄)alkyl substituted by any of the previously described cycloalkyl,cycloalkenyl, aryl or heterocyclic groups; any of the previous groupsinterrupted and/or substituted by one or more heteroatoms, e.g. so as tocontain one or more ether, thioether, amino, amido, carbonyl orthiocarbonyl groups; or any of the previous groups carrying one or moresubstituents which may, for example, be selected from hydroxy, mercapto,amino, halo, nitro, azido, carboxy, cyano and isothiocyanato, or anyother substituent compatible with the boronic acid residue. Indeed ingeneral in the compounds of the invention, unless otherwise specified,alkyl, alkenyl and alkenylene moieties will contain up to 6 carbon atomsand cyclic groups will have 5 to 7 membered rings.

Although the absorption maxima of dyes derived from compounds of formulaIX are normally below 700 nm, some derivatives may also possessabsorption in the near infrared area. Examples of such compounds arethose derived from compounds of formula IX where two or more of thephenyl groups are further linked, e.g. by a bond or a bridging group, toform a further at least five membered ring. Examples of such dyesinclude those in which the label is derived from a compound of formula X##STR8##

where R₁ and A are as defined above. Absorption maxima for such dyes andthe size of the bathochromic shift depends on the auxochrome groupspresent in such derivatives. Compared to Crystal Violet (A/A/A =NMe₂/NMe₂ /NMe₂) and Malachite Gren (A/A/A=NMe₂ /NMe₂ /H), such bridgingdisplaces the longest absorption band to longer wavelength, resulting inabsorption at 850 nm and 955 nm, respectively. Even greater bathochromicshifts can be produced using acetylenic analogues of triphenylmethinedyes of formula XI ##STR9##

(where X is a bond or a linking atom or group, e.g. oxygen, nitrogen,sulphur or carbon substituted according to valence demands, e.g. withprotons or organic groups). Thus X may be C(R₁)₂ where R₁ is for examplean alkyl group).

In formula XI, the resonance system is further increased (by greaterdelocalization of the positive charge) by the triple bond as well as thebridging of the phenyl rings.

These derivatives often possess several absorption bands in the nearinfrared wavelength area and distinct absorption bands are seen around1000 nm.

Another particularly useful category of compounds according to theinvention includes those in which V is derived from a cyanine ormercyanine dye, e.g. as represented by formulae XII and XIIIrespectively ##STR10##

and the corresponding alternative resonance structures. In the aboveformulae A' and A" represent quaternized heterocyclic bases orheterocyclic aromatic groups containing ring oxygen and/or sulphuratoms; D represents a ketomethylene-derived nucleus; R is ashereinbefore defined; and m' is an integer, e.g. of 1-3, conveniently 2or 3.

The analogous compounds wherein the terminal A' and A" rings areattached at carbons adjacent ring oxygen or sulphur atoms instead, i.e.the structural isomers of formulae XIV and XV are also suitable##STR11##

(where R, m', A', A" and D are as defined above and Y is O or S).

Examples of dyes of this type include those of formula XVI ##STR12##

in which m' and R are as hereinbefore defined, X₂ represents aheteroatom such as oxygen or sulphur or an optionally mono- ordi-substituted methylene group, and each R₂ independently represents ahydrogen atom, an organic group (e.g. as hereinbefore described for R₁)or a (preferably water-solubilizing) substituting group, or adjacent R₂groups may together with the carbon atoms to which they are attachedform fused monocyclic or polycyclic ring systems. Water-solubilityenhancing substituents are desirably present as one or more of R₂, onepreferred such substituent being carboxymethyl, which will conjugate toamine-containing molecules such as m-aminophenyl boronic acid withlittle or no change in the absorption characteristics of the cyaninechromophore. The extensive conjugation seen in these molecules canincrease their chemical/photochemical instability, but appropriate dyedesign, for instance incorporating the polymethine chain into one ormore cyclic structures is possible. Examples of such dyes are{4-[7-(2-phenyl-4H-1-benzothiopyran-4-ylidene)-4-chloro-3,5-trimethylene-1,3,5-heptatrienyl]-2-phenyl-1-benzothiopyriliumperchlorate}, and3,3'-diethyl-9,11,15,17-dineopentylene-thiapentacarbocyanineperchlorate.

The labelled boronic acid conjugates of the invention can be prepared byreacting an appropriately functionalized reporter molecule having thedesired reporter characteristics, e.g. spectral characteristics, tocouple it to the desired phenylboronic acid residue as described above.

The labelled conjugates of the invention may be used in diagnostic testprocedures, e.g. the glycated blood protein assays of WO-92/08722 andWO-90/13818, and this use and diagnostic kits therefor comprise furtheraspects of the invention.

Thus viewed from a further aspect the invention provides the use of alabelled boronic acid compound of formula III or a salt thereof in anassay procedure, suitably a diagnostic assay, especially a blood assay.

Viewed from a yet further aspect the invention also provides an assaykit comprising a compound of formula III or a salt thereof, means forcontacting a cis-diol containing fluid sample with said compound, meansfor separating labelled, cis-diol conjugates thus formed and optionallymeans for assessing said separated conjugates.

Thus the assay kit may conveniently comprise a solution (e.g. a zinccontaining aqueous solution buffered to pH 6.5 to 8, preferably 7 to7.5) of a compound of formula III or salt thereof, a mixing vessel and aporous web (e.g. filter paper) capable of retaining the labelledcis-diol conjugates. Optional assessment means may conveniently comprisea spectrophotometer appropriately arranged to detect light emitted by(or modified by) the label.

The following non-limiting Examples are provided to illustrate theinvention further:

Example 1: 4-Carboxy-phenylboronic acid (CPBA) ##STR13##

4-Methyl-boronic acid (5g, 0.037 mol) was dissolved in 300 ml 0.25 MNaOH using a magnetic stirrer. After 10 minutes, a suspension of KMnO₄(12.3 g, 0.078 mol in 130 ml water) was added and the solution was leftto react overnight (approx. 12 hours) at ambient temperature. Thesolution was then heated to 60° C. and hot-filtered and the filtersubsequently washed with 2×50 ml hot water. The filtrate was thenreduced to 1/3 volume, heated to 60° C. and finally made slightly acidicby adding concentrated hydrochloric acid before cooling. Whiteneedle-like crystals were collected in 75% yield.

Example 2:4-Benzotriazolyloxycarbonyl-phenylboronic acid (HBT-CPBA)##STR14##

To a solution of 4-carboxy-phenylboronic acid (CPBA, Example 1, 2 g,0.0121 mol) in N,N-dimethylformamide (DMF) was added1-hydroxybenzotriazole (HBT, 2.04 g, 0.0151 mol) andN,N'-dicyclohexyl-carbodiimide (DCC, 4.98 g, 0.024 mol) both dissolvedin DMF (the total volume was 170 ml DMF). The solution was left to reactovernight (approx. 12 hours) under a nitrogen atomosphere and at ambienttemperature, and then filtered to remove the insoluble urea byproduct(DCU). The title compound was used in subsequent reactions withoutfurther purification.

Example 3: 4-Succinimidyloxycarbonyl-phenylboronic acid (NHS-CPBA)##STR15##

4-Carboxyphenyl-boronic acid (CPBA, Example 1, 0.25 g, 1.51 mmol) andN-hydroxysuccinimide (0.26 g, 2.26 mmol) were dissolved in 8.5 ml DMFusing a magnetic stirrer. 20 ml of dichloromethane (CH₂ Cl₂) was thenadded, and the solution was stirred for 10 minutes.N,N'-dicyclohexyl-carbodiimide (DCC, 0.93 g, 4.52 mmol) in 3 ml CH₂ Clwas then added. The solution was left to react under a nitrogenatmosphere overnight (approx. 12 hours) at ambient temperature, and thenfiltered to remove the insoluble urea byproduct (DCU). The titlecompound was used in subsequent reactions without further purification.

Example 4:4-((3-Amino-2-hydroxypropyl)aminocarbony1)-phenylboronic acid(CPBA-DAPOL) ##STR16##

A newly filtered solution of HBT-CPBA (Example 2, 0.0121 mol) was addedthrough a dropping funnel to a solution of 1,3-diamino-2-propanol(DAPOL, 1.09 g, 0.0121 mol) in 15 ml pyridine. The mixture was stirredunder nitrogen and left to react at ambient temperature overnight(approx. 12 hours). The product was isolated and purified bychromatography on silica gel eluting with methanol:NH₃ (aq), 1:0.5. Thetitle compound was isolated as matt white plate-like crystals.

Example 5: 4-((3-Amino-2-hydroxypropy1)aminocarbonyl)-phenylboronic acid(CPBA-DAPOL)

The procedure of Example 4 was repeated using a newly filtered solutionof NHS-CPBA (Example 3, 0.0121 mol) instead of HBT-CPBA.

Example 6:4-((3,6-Diaza-hex-1-yl)aminocarbonyl)-phenylboronic acid(CPBA-BAEA) ##STR17##

A newly filtered solution of HBT-CPBA (Example 2, 0.00121 mol) was addedthrough a dropping funnel to a solution of bis-(2-aminoethyl)-amine(BAEA, 0.00133 mol) in 1.5 ml pyridine. The mixture was stirred undernitrogen and left to react overnight (approx. 12 hours) at ambienttemperature. The title compound was isolated and purified bychromatography on silica.

Example 7: 4-((3,6-Diaza-hex-1-yl)aminocarbonyl)phenylboronic acid(CPBA-BAEA)

The procedure of Example 6 was repeated using a newly filtered solutionof NHS-CPBA (Example 3, 0.00121 mol) instead of HBT-CPBA.

Example 8: 4-(5-Amino-3-oxapent-1-yl)aminocarbonyl)phenylboronic acid(CPBA-BAEE) ##STR18##

A newly filtered solution of HBT-CPBA (Example 2, 0.00121 mol) was addedthrough a dropping funnel to a solution of bis-(2-aminoethyl)-ether(BAEE, 0.00106 mol) in 10 ml of 0.4M sodium-bicarbonate buffer, pH 8.5.The mixture was stirred and left to react overnight (approx. 12 hours)at ambient temperature. The reaction mixture was evaporated to drynessand the title compound isolated and purified by chromatography onsilica.

Example 9: 4-(5-Amino-3-oxapent-1-yl)aminocarbonyl)phenylboronic acid(CPBA-BAEE)

After removal of dichloromethane by rotary evaporation from a CH₂ Cl₂/DMF solution of NHS-CPBA (Example 3, 0.0013 mol), the resultingNHS-CPBA in solution in 8 ml DMF was filtered and added to a solution ofbis-(2-aminoethyl)-ether (BAEE, 0.00106 mol) in 10 ml of 0.4Msodium-bicarbonate buffer, pH 8.5. The mixture was stirred and left toreact overnight (approx. 12 hours) at ambient temperature. The reactionmixture was evaporated to dryness and the title compound isolated andpurified by chromatography on silica.

Example 10: 4-((2-Aminoethyl)aminocarbonyl)-phenylboronic acid(CPBA-EDA) ##STR19##

A newly filtered solution of HBT-CPBA (Example 2, 0.00121 mol) was addedthrough a dropping funnel to a solution of 1,2-ethylenediamine (EDA,0.00145 mol) in 1 ml pyridine. The mixture was stirred under nitrogenand left to react overnight (approx. 12 hours) at ambient temperature.The title compound was isolated and purified by chromatography onsilica.

Example 11: 4-((2-Aminoethyl)aminocarbonyl)-phenylboronic acid(CPBA-EDA)

The procedure of Example 10 was repeated using a newly filtered solutionof NHS-CPBA (Example 3, 0.00121 mol) instead of HBT-CPBA.

Example 12: 4-((3-Aminopropyl)aminocarbonyl)phenylboronic acid(CPBA-DAP) ##STR20##

A newly filtered solution of HBT-CPBA (Example 2, 0.00106 mol in 1 mlDMF) was added through a dropping funnel to a solution of1,3-diamino-propane (DAP, 0.00127 mol) in 1 ml pyridine. The mixture wasstirred under nitrogen and left to react overnight (approx. 12 hours) atambient temperature. The title compound was isolated and purified bychromatography on silica.

Example 13: 4-((3-Aminopropyl)aminocarbonyl)phenylboronic acid(CPBA-DAP)

The procedure of Example 12 was repeated using a newly filtered solutionof NHS-CPBA (Example 3, 0.0121 mol) instead of HBT-CPBA.

Example 14: 4-((6-Aminohexyl)aminocarbonyl)-phenylboronic acid(CPBA-DAH) ##STR21##

A newly filtered solution of HBT-CPA (Example 2, 0.00106 mol in 10 mlDMF) was added through a dropping funnel to a solution of1,6-diamino-hexane (DAP, 0.00127 mol) in 1 ml pyridine. The mixture wasstirred under nitrogen and left to react overnight (approx. 12 hours) atambient temperature. The title compound was isolated and purified bychromatography on silica.

Example 15: 4-((6-Aminohexyl)aminocarbony1)-phenylboronic acid(CPBA-DAH)

The procedure of Example 14 was repeated using a newly filtered solutionof NHS-CPBA (Example 3, 0.0121 mol) instead of HBT-CPBA.

Example 16: 4-((4-Amino-4-carboxy-butyl)aminocarbonyl)phenylboronic acid(CPBA-ornithine) ##STR22##

A newly filtered solution of HBT-CPBA (Example 2, 0.00042 mol in 4 mlDMF) was added through a dropping funnel to a solution ofNδ-t-Boc-L-ornithine (Boc-Orn, 0.0005 mol) in 2 ml of 0.4M sodiumbicarbonate buffer, pH 8.5/10 ml DMF. The mixture was stirred and leftto react overnight (approx. 12 hours) at ambient temperature. Thereaction mixture was evaporated to dryness and the residue dissolved in10 ml DMF. 5 ml of a solution comprising CHCl₃ /trifluoroacetic acid(50/50) was then added to remove the amine-protecting Boc-group, and thedeprotection was followed by TLC-analysis on silica in methanol:NH₃(aq)-3:1. When deprotection was completed (<1 hour) the resulting titlecompound was isolated and purified by chromatography on silica.

Example 17: 4-((6-Aminohexyl)aminocarbonyl)-phenylboronic acid(CPBA-ornithine)

After removal of dichloromethane by rotary evaporation from a CH₂ Cl₂/DMF-solution of NHS-CPBA (Example 3, 0.0010 mol), NHS-CPBA in solutionin 5 ml DMF was filtered and added to a solution of Nδ-t-Boc-L-ornithine(Boc-Orn, 0.0005 mol) in 2 ml of 0.4M sodium bicarbonate buffer, pH8.5/10 ml DMF. The mixture was stirred and left to react overnight(approx. 12 hours) at ambient temperature. The reaction mixture wasevaporated to dryness and the residue dissolved in 10 ml DMF. 5 ml of asolution comprising CHCls/trifluoroacetic acid (50/50) was then added toremove the amine-protecting Boc-group, and the deprotection was followedby TLC-analysis on silica in methanol:NH₃ (aq)--3:1. When deprotectionwas completed (<1 hour) the resulting title compound was isolated andpurified by chromatography on silica.

Example 18: Xylene-cyanole-phenylboronic acid conjugates

(A) XC-DAPOL-CPBA ##STR23##

Purified CPBA-DAPOL (Example 4 or 5, 0.05 g, 0.000210 mol) was dissolvedin 50 μl methanol before 1 ml of DMF and 10.5 ml of 0.1Msodium-bicarbonate buffer, pH 8.6 were added. Under constant stirringusing a magnetic stirrer, Xylene-cyanole-SO₂ Cl (139 mg, 0.00026 mol in5 ml of methylcyanide) was added over a period of 5 minutes. Thereaction mixture was left to react overnight (approx. 12 hours) atambient temperature. The product, XC-DAPOL-CPBA, was purified twice onsilica using pure methanol as solvent. The isolated dye-boronic acidderivative possess an absorption maximum at 616 nm, extinctioncoefficient =70 000 1/mol,cm.

(B) to (G) XC-"spacer"-CPBA

The procedure described for Example 18(A) is repeated using thecompounds of Examples 6/7, 8/9, 10,11, 12/13, 14/15 and 16/17 instead ofthat of Examples 4/5.

Example 19 (Comparative)

Deboronation of a Xylene Cyanole-3-aminophenylboronic acid-conjugate

Chromatographically pure (>96%) Xylene Cyanole-3-aminophenylboronicacid-conjugate (synthesized and isolated as described in Example 2 ofWO-92/08722) was dissolved in DMSO and then diluted to a finalconcentration of 1.9×10⁻⁴ M in 100 mM sodium-bicarbonate-buffer/15%DMSO, pH 9.7 before being stored for 3 days at ambient temperature.After storage, the solution was chromatographed using reversed phasechromatography (RPC) on ClS-bonded silica. Two blue-dyed main fractionswere isolated. On analytical RPC using a 20 mM ammonium acetate,pH7.0/methanol-gradient, the isolated dyed compounds eluted differentlythan the fresh Xylene Cyanole-3-aminophenyl boronic acid-conjugate.Elemental analysis showed no boron in the isolated compounds. Thesefindings were confirmed by the results obtained using the methoddescribed in Example 7 of WO-92/08722. No signal response as a functionof blood hemoglobin glycation was found, indicating the absence ofactive conjugate.

Example 20 (Comparative)

Deboronation of a Xylene Cyanole-3-aminophenylboronic acid-conjugate

Chromatographically pure (>96%) Xylene Cyanole-3-aminophenylboronicacid-conjugate (synthesized and isolated as described in Example 2 ofWO-92/08722) was dissolved in DMSO and then diluted to a finalconcentration of 1.9×10⁴ M using a buffer solution comprising 100 mMglycylglycine, 25 mM zinc-chloride, 0.07% Triton X-100, 10% DMSO, pH9.7. The solution was stored at 4° C. for 14 days, and thenchromatographed using reversed phase chromatography (RPC) on C18-bondedsilica. Three blue-dyed compounds were isolated. Two of the isolatedcompounds (representing >70% of the blue dye intensity) eluteddifferently than the fresh Xylene Cyanole-3-aminophenyl boronicacid-conjugate on analytical RPC using a 20 mM ammonium acetate, pH7.0/methanol-gradient. Elemental analysis showed no boron in thesecompounds. These findings were confirmed by the results obtained usingthe method described in Example 7 of W0-92/08722. No signal response asa function of blood hemoglobin glycation was found, indicating theabsence of active conjugate in the isolated blue-dyed derivative.

Example 21

Stability of Xylene Cyanole-3-aminophenylboronic acid and thecorresponding DAPOL-CPBA-conjugate stored in a neutral solution at 30°C./37° C.

Two identical solutions of XC dye-boronic acid-conjugates were madeusing for one Xylene Cyanole-3-aminophenylboronic acid produced andisolated as described in Example 2 of WO-92/08722 and for the other thecorresponding Xylene Cyanole-DAPOL-CPBA conjugate produced and isolatedas described in Example 18(A).

Solution I comrised:

Xylene Cyanole-3-aminophenyl boronic acid (XC-APBA, 2.3×10⁻⁴ M), 100 mMglycinamide, 25mM zinc-chloride, 0.07% Triton X-100, 16% formamide, pH7.5.

Solution II comprised:

Xylene Cyanole-DAPOL-CPBA (XC-DAPOL-CPBA, 2.3×10⁻⁴ M), 100 mMglycinamide, 25 mM zinc-chloride, 0.07% Triton X-100, 16% formamide, pH7.5.

Both solutions were stored at 30° C. for 14 days.

During storage the stability of these solutions was investigated byusing them as working solutions of "dye-boronic acid conjugate" in thedetermination of glycohemoglobin as described below. Reducedavailability of active conjugate, either resulting from deboronation orother degradative reactions on the dye-boronic acid conjugate duringstorage, was displayed as a malfunction of the assay in thedetermination of glycohemoglobin.

Measurement of glycohemoglobin in whole blood using XyleneCyanole-boronic acid conjugate

To test the effect of storage on solutions I and II, the incubatedsolutions were used separately to determine glycohemoglobin in wholeblood samples, repeating the procedure at different times of storage.2.5 μl of a whole blood sample was mixed with 150 μl of solution I orII, respectively. The whole blood sample was hemolyzed and a precipitatewas formed. The precipitated hemoglobin was separated by filtration, andits reflectance measured at 685 and 470 nm by a reflectometer. The ratioof the reflectances at 685 and 470 nm was calculated, and the percentageof glycohaemoglobin to total haemoglobin was determined from acalibration curve obtained using standard solutions of knownconcentrations of haemoglobin and glycohaemoglobin.

Blood samples with known glycohemoglobin values were used in this study,and the resulting standard curve was compared with the curve obtainedwith corresponding freshly made dye-boronic acid conjugate solutions. Ifthe concentration of active conjugate is reduced in the dye-boronic acidconjugate solution, a declining slope is observed. The effect on theslope was used as a measure of conjugate stability.

Already after storage for 6 days at 30° C., solution I showed asignificant change in signal response as a function of blood hemoglobinglycation, whereas no significant negative effects were seen usingsolution II. Further incubation for 6 days resulted in a distinct dropin signal response using solution I, but still no detrimental effectswere seen using solution II. After further incubation for 5 days(total=17 days), now at 37° C., still no negative effects were seenusing solution II. At this time, the XC-APBA-conjugate in solution Ishowed almost no functionality. By visual inspection of the solutionsduring storage, it was also easy to observe a distinct decrease in colorintensity in solution I as a function of time. The observed differencesbetween solution I and II demonstrate the differences in stability ofthe two tested Xylene Cyanole (XC)-boronic acid conjugates.

Compared with the corresponding XC-APBA-conjugate, theXC-DAPOL-CPBA-conjugate showed superior stability under all conditionstested.

Example 22: Phthalocyanine-labelled boronic acid conjugate

Chloroaluminium phthalocyanine tetrasulphonate (100 mg, 1.12×10⁻⁴ mol)is dried overnight at 100° C. Phosphorus oxychloride (410 μl, 4.48×10⁻³mol) is added and the mixture is incubated at ambient temperature underexclusion of moisture. After 24 hours at this temperature, unreactedphosphorus oxychloride is removed by bulb-bulb distillation.

The remaining solid phthalocyanine sulphonyl chloride is washed withchloroform and dried under reduced pressure and exclusion of moisture.500 μl dry DMF is added to solubilize the phthalocyaninesulphonylchloride. Immediately thereafter 3 ml of 0.3 M NaHCO₃ solutionpH 8.5, containing 1.3*10⁻⁴ mol CPBA-DAPOL (prepared as described inExample 4) is added, and the reaction mixture is left to react for aminimum of 2 hours at constant pH. The boronic acid conjugates areisolated by reversed phase chromatography, mainly in the form ofmono(phenyl boronic acid)--functionalized dye.

Example 23: Cyanine dye--boronic acid conjugate

Phosphorus oxychloride (470 μl, 5.13×10⁻³ mol) is added to the cyaninedye2-[7-[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benz[e]indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)-1H-benz[e]indoliumhydroxide, inner salt, sodium salt (commercially known as IR 125 orindocyanine green) (100 mg, 1.29×104 mol) and the resulting mixture isincubated at ambient temperature under exclusion of moisture for 24hours. Unreacted phosphorus oxychloride is removed by bulb-bulbdistillation. The IR-125 sulphonyl chloride is extracted intochloroform, and the extract is washed five times with 1 ml portions ofcold water and then evaporated to dryness under reduced pressure andexclusion of moisture.

The resulting solid material is immediately solubilized in DMF (500 μl),to which solution is added 3 ml of 0.3 M NaHCO₃ solution, pH 8.5,containing 1.1 , 10⁻⁴ mol CPBA-DAPOL (prepared as described in Example4) and the reaction mixture is left to react for a minimum of 2 hours atconstant pH. The boronic acid conjugates are isolated by reversed phasechromatography, mainly in the form of mono(phenyl boronicacid)-functionalized dye.

Example 24: Cyanine dye--boronic acid conjugate

To the reactive succinimidyl ester-cyanine dye Cy5.18 (1.3×10⁻⁷ mol) areadded 1.5 , 10⁻⁴ mol CPBA-DAPOL (prepared as described in Example 4) in0.5 ml 0.1M sodium carbonate/sodium bicarbonate buffer (pH 9.3), withsubsequent thorough mixing. The mixture is incubated under constantstirring at ambient temperature for 4 hours, and the phenyl boronic acidconjugate finally isolated by reversed phase chromatography.

Example 25: Cyanine--boronic acid-conjugate

The methyl ester of the cyanine dye commercially known as IR 132 indioxane, is transformed into its dicarboxylic acid analogue by titrationwith aqueous hydroxide (LiOH) using equimolar amounts of ester and base.

To 100 mg of the carboxylic acid containing cyanine dye (1.1E-4 mol) isadded 450 μl of phosphorus oxychloride (5.13E-3 mol) and the mixture isincubated at ambient temperature with the exclusion of moisture for 24hours. The dark solution is poured over crushed ice, left for 5 minutesand the IR-132-carboxylic acid-chloride extracted into 40 ml ofchloroform.

The chloroform extract is washed five times with 5 ml of ice-cold waterand finally stored over anhydrous Na₂ SO₄. Before being used further thedry chloroform-acid chloride extract is evaporated to dryness underreduced pressure and with the exclusion of moisture. The solid materialis dissolved in 2 ml of dry methylene chloride and then 1.5*10⁻⁴ mol ofCPBA-DAPOL (prepared as described in Example 4) dissolved in 3 ml of 0.3M NaHCO₃ solution, pH 8.5, is added. The reaction mixture is left toreact for a minimum of 2 hours at constant pH and the boronic acidconjugates are isolated by reverse phase chromatography.

We claim:
 1. Labelled boronic acid compound of formula ##STR24##(wherein V is a reporter moiety; W² is a bond or an organic linkermoiety;W¹ is a *SO₂ NR², *CONR² or *CH₂ N⊕R² ₂ group bound at the*-marked atom to the phenyl ring; R¹ is hydrogen or an electronwithdrawing substituent group; and each R² independently is hydrogen oran optionally hydroxylated and optionally C₁₋₆ -alkoxylated C₁₋₆ -alkylgroup) and salts thereof.
 2. Compounds as claimed in claim 1 wherein W¹is a SO₂ NR² or CONR² group attached to the phenyl ring at the4-position thereof.
 3. Compounds as claimed in claim 1 wherein W¹ is aSO₂ NR² or CONR² group attached to the. phenyl ring at the 2, 3, 5 or6-position thereof.
 4. Compounds as claimed in claim 1 wherein W¹ is aCH₂ N⊕R₂ ² group attached to the phenyl ring at the 3 or 5 positionthereof.
 5. Compounds as claimed in claim 1 wherein W¹ is a CH₂ N⊕R₂ ²group attached to the phenyl ring at the 2 4 or 6-position thereof. 6.Compounds as claimed in claim 1 wherein R¹ denotes an SO₂ H, CO₂ H orNO₂ group attached at the 3 or 5 position of the phenyl ring. 7.Compounds as claimed in claim 1 wherein R¹ denotes an SO₂ H, CO₂ H orNO₂ group attached at the 2, 4 or 6-positions of the phenyl ring. 8.Compounds as claimed in claim 1 wherein W² is a group of formula IV

    --X.sup.1 [(CHR.sup.3).sub.n X.sup.2 ].sub.m --(CHR.sup.3).sub.n --(IV)

(wherein each n is independently an integer having a value of 1 to 6, mis 0 or an integer having a value of 1 to 5, X¹ is a bond, an oxygen orsulphur atom, a NR² group, or a carboxy or carbonyl group, each X² isindependently an oxygen or sulphur atom or an NR² group, R² is asdefined in claim 1, and each R³ independently is hydrogen, hydroxy,formyl, carboxy, or optionally hydroxylated and/or C₁₋₆ -alkoxylatedC₁₋₆ -alkyl, or a group CHR₃ represents a carbonyl group).
 9. Compoundsas claimed in claim 8 wherein W² is selected from the group consistingof:--NH--CH₂ --CHOH--CH₂ -- --NH--CH₂ CH₂ --NH--CH₂ CH₂ -- --NH--CH₂ CH₂--O--CH₂ CH₂ -- --NH--CH₂ CH₂ -- --NH--CH₂ CH₂ CH₂ -- --NH--CH₂ CH₂ CH₂CH₂ -- --NH--CH₂ CH₂ CH₂ CH(COOH)-- NH--CH₂ CHOH-- --NH--CH₂ CH(COOH)----NH--CH₂ --NH--CH₂ -- --NH--CH₂ CH₂ CH₂ CH₂ CH₂ CH₂ -- --NH--CH₂ CH₂CH₂ CH₂ CH₂ -- --NH--CH₂ --O-CH₂ -- --NH--CH₂ --O--CH₂ --O--CH₂ ----NH--CH₂ CH₂ --O--CH₂ CH₂ --O--CH₂ CH₂ -- --NH--CH(COOH)CH₂ CH₂ ----NH--CH(COOH)CH₂ CH₂ CH₂ CH₂ --NH--CH₂ CH(COOH)CH(COOH)-- --NH--CH₂CHOHCHOH-- and --NH CH₂ CH(CHO)CH(CHO)-- groups.
 10. Compounds asclaimed in claim 1 wherein V is a chromophore or fluorophore. 11.Compounds as claimed in claim 10 wherein V has an absorption maximum inthe range 600-1000 nm.
 12. Compounds as claimed in claim 10 wherein V isan azine, triarylmethine, phthalocyanine or cyanine dye.
 13. Compoundsas claimed in claim 12 wherein V is a xylene-cyanole dye.
 14. Compoundsas claimed in claim 1 being compounds of formula (V) ##STR25## where Vand R² are as defined in claim 1, X³ is a CO or SO₂ group, and W² is agroup of formula IV

    --X.sup.1 [(CHR.sup.3).sub.n X.sup.2 ].sub.m --(CHR.sup.3).sub.n --(IV)

wherein each n is independently an integer having a value of 1 to 6; mis 0 or an integer having a value of 1 to 5; X¹ is a bond, an oxygen orsulfur atom, a NR² group, or a carboxy or carbonyl group, each X² isindependently an oxygen or sulphur atom or an NR² group, R² is asdefined in claim 1, and each R³ independently is hydrogen, hydroxy,formyl, carboxy, or optionally hydroxylated and/or C₁₋₆ -alkoxylatedC₁₋₆ -alkyl, or a group CHR³ represents a carbonyl group.
 15. Compoundsas claimed in claim 1 being compounds of formula VI ##STR26## (where V,R¹ and W² are as defined in claim 1, and W³ is NHCO or SO₂ NH) and saltsthereof.
 16. Compounds as claimed in claim 1 being compounds of formulaVII ##STR27## (where V, W² and R² are as defined in claim 1) and saltsthereof.
 17. Compound of formula VIII ##STR28## (where X², R¹, R³, n mand W¹ are as defined in claim 8 and X^(1*) is a reactive functionalgroup) and salts thereof.